Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):
- EV2.16 bisulfite
- EV2.20 bisulfite
- EV2.22 bisulfite
- EV2.24 bisulfite
- EV2.28 bisulfite
- EV2.29 bisulfite
- EV2.32 bisulfite
- EV2.33 bisulfite
DNA needed to be diluted. Diluted according to this sheet provided by Mac:
NOTE: EV2.28 didn’t have sufficient DNA left to prepare the dilution according to Mac’s sheet. Instead, the remaining volume ofEv2.28 bisulfite DNA (0.5uL) was diluted in a total volume of 2.5uL to maintain the same dilution ratio.
Master mix calcs are here: 20140828 – PCR Mac Bisulfite Samples
Primers used were:
- 1. 95C – 10mins
- 2. 94C – 30s
- 3. 56C – 30s
- 4. 72C – 30s
- 5. Repeat steps 2 – 5 44 more times
- 6. 72C – 10mins
Ladder used is O’GeneRuler 100bp DNA Ladder (ThermoFisher).
According to Mac, the expected band size is ~300bp. However, all samples are running at ~150bp. Mac is confused and does not know what to do.
*UPDATE 20140902* – Realized I used the wrong forward primer! Will repeat PCR with correct primer. Wonder if Mac did the same thing…